A variety of inherited and acquired diseases are associated with genetic variations such as point mutations, deletions and insertions. Some of these variants are directly associated with the presence of disease, while others correlate with disease risk and/or prognosis. There are more than 500 human genetic diseases which result from mutations in single genes. These include cystic fibrosis, muscular dystrophy, .alpha.1-antitrypsin deficiency, phenylketonuria, sickle cell anaemia or trait, and various other haemoglobinopathies. Furthermore, individuals with increased susceptibility to several common polygenic conditions, such as atherosclerotic heart disease, have been shown to have an association with the inheritance of a particular DNA sequence polymorphism. Cancer is thought to develop due the accumulation of genetic lesions in genes involved in cellular proliferation or differentiation. The ras proto-oncogenes, K-ras, N-ras, and H-ras, and the p53 tumor suppressor gene are examples of genes which are frequently mutated in human cancers. Specific mutations in these genes result in an increase in transforming potential. Genetic analysis is likely to become routine in the clinic for assessing disease risk, diagnosis of disease, predicting a patient's prognosis or response to therapy, and for monitoring a patient's progress. The introduction of such genetic tests depends on the development of simple, inexpensive, and rapid assays for genetic variations.
Due to increasing interest in the development of such tests a number references have been published in this area, these include, Abravaya, K., Carrino, J. J., Muldoon, S. and Lee, H. H. (1995) Detection of point mutations with a modified ligase chain reaction (Gap-LCR). Nucleic Acids Research 23, 675-682; Barany, F. (1991) Genetic disease detection and DNA amplification using cloned thermostable ligase. Proc. Natl. Acad. Sci. 88, 189-193; Belgrader, P., Marino, M. M., Lubin, M. and Barany, F. (1996) A multiplex PCR-Ligase detection reaction assay for human identity testing. Genome Science and Technology 1, 77-87; and Eggerding, F. A. (1995) A one-step coupled amplification and oligonucleotide ligation procedure for multiplex genetic typing. PCR Methods and Applications 4, 337-345.
In U.S. Pat. No. 5,593,840 there is disclosed a method of detecting a particular nucleic acid sequence. The method disclosed in this patent is said to be based on the discovery that certain aspects of polymerase chain reaction (PCR) and ligase chain reaction (LCR) can be used in combination to detect and amplify a target nucleic acid sequence. This method involves the use of three primers, with the third primer being complementary to at least a portion of the 5' end of the first primer. It is an essential feature of this method that the position of the third primer complementary to the base at the 5' end of the first primer contains a modification such as to substantially avoid strand displacement by polymerase.
Further information regarding PCR and LCR can be found in U.S. Pat. Nos. 4,683,202, 4,683,195, 4,800,159, 4,965,188, 5,176,995, EP 0 320 308 and EP 0 439 182 and the disclosure of these references is included herein by reference.